Arylpiperazines and arylpiperidines and their use as metalloproteinase inhibiting agents

ABSTRACT

Compounds of the formula I  
                 
 
     useful as metalloproteinase inhibitors, especially as inhibitors of MMP 13.

[0001] The present invention relates to compounds useful in theinhibition of metalloproteinases and in particular to pharmaceuticalcompositions comprising these, as well as their use. In particular, thecompounds of this invention are inhibitors of matrix metalloproteinase13 (MMP13), known also as collagenase 3.

[0002] Metalloproteinases are a superfamily of proteinases (enzymes)whose numbers in recent years have increased dramatically. Based onstructural and functional considerations these enzymes have beenclassified into families and subfamilies as described in N. M. Hooper(1994) FEBS Letters 354:1-6. Examples of metalloproteinases include thematrix metalloproteinases (MMPs); the reprolysin or adamalysin or MDCfamily which includes the secretases and sheddases such as TNFconverting enzymes (ADAM10 and TACE); the astacin family which includeenzymes such as procollagen processing proteinase (PCP); and othermetalloproteinases such as aggrecanase, the endothelin converting enzymefamily and the angiotensin converting enzyme family.

[0003] Metalloproteinases are believed to be important in a plethora ofphysiological disease processes that involve tissue remodelling such asembryonic development, bone formation and uterine remodelling duringmenstruation. This is based on the ability of the metalloproteinases tocleave a broad range of matrix substrates such as collagen, proteoglycanand fibronectin. Metalloproteinases are also believed to be important inthe processing, or secretion, of biological important cell mediators,such as tumour necrosis factor (TNF); and the post translationalproteolysis processing, or shedding, of biologically important membraneproteins, such as the low affinity IgE receptor CD23 (for a morecomplete list see N. M. Hooper et al., (1997) Biochem J. 321:265-279).

[0004] Metalloproteinases have been associated with many diseases orconditions. Inhibition of the activity of one or more metalloproteinasesmay well be of benefit in these diseases or conditions, for example:various inflammatory and allergic diseases such as, inflammation of thejoint (especially rheumatoid arthritis, osteoarthritis and gout),inflammation of the gastro-intestinal tract (especially inflammatorybowel disease, ulcerative colitis and gastritis), inflammation of theskin (especially psoriasis, eczema, dermatitis); in tumour metastasis orinvasion; in disease associated with uncontrolled degradation of theextracellular matrix such as osteoarthritis; in bone resorptive disease(such as osteoporosis and Paget's disease); in diseases associated withaberrant angiogenesis; the enhanced collagen remodelling associated withdiabetes, periodontal disease (such as gingivitis), corneal ulceration,ulceration of the skin, post-operative conditions (such as colonicanastomosis) and dermal wound healing; demyelinating diseases of thecentral and peripheral nervous systems (such as multiple sclerosis);Alzheimer's disease; extracellular matrix remodelling observed incardiovascular diseases such as restenosis and atheroscelerosis; andchronic obstructive pulmonary diseases, COPD (for example, the role ofMMPs such as MMP12 is discussed in Anderson & Shinagawa, 1999, CurrentOpinion in Anti-inflammatory and Immunomodulatory Investigational Drugs,1(1): 29-38).

[0005] The matrix metalloproteinases (is) are a family ofstructurally-related zinc-containing endopeptidases which mediate thebreakdown of connective tissue macro-molecules. The mammalian MMP familyis composed of at least twenty enzymes, classically divided into foursub-groups based on substrate specificity and domain structure[Alexander & Werb (1991) in Hay, E. D. ed. “Cell Biology of theExtracellular Matrix”, New York, Plenum Press, 255-302; Murphy &Reynolds (1993) in Royce, P. M. & Steinman, B. eds. “Connective Tissueand its Heritable Disorders”, New York, Wiley-Liss Inc., 287-316;Birkedal-Hansen (1995) Curr. Opin. Cell Biol. 7:728-7351. The sub-groupsare the collagenases (such as MMP1, MMP8, MMP13), the stromelysins (suchas MMP3, MMP10, MMP11), the gelatinases (such as MMP2, MMP9) and themembrane-type MMPs (such as MMP14, MMP15, MMP16, MMP17). Enzyme activityis normally regulated in vivo by tissue inhibitors of metalloproteinases(TIMPs).

[0006] Because of their central role in re-modelling connective tissue,both as part of normal physiological growth and repair and as part ofdisease processes, there has been substantial interest in these proteinsas targets for therapeutic intervention in a wide range of degenerativeand inflammatory diseases, such as arthritis, atherosclerosis, andcancer (Whittaker et al (1999) Chem. Rev. 99:2735-2776).

[0007] A number of MMP inhibitor compounds are known and some are beingdeveloped for pharmaceutical uses (see for example the review by Beckett& Whittaker (1998) Exp. Opin. Ther. Patents, 8(3):259-282). Differentclasses of compounds may have different degrees of potency andselectivity for inhibiting various MMPs. Whittaker M. et al (1999, Chem.Rev. 99:2735-2776) review a wide range of known MMP inhibitor compounds.They state that an effective MMP inhibitor requires a zinc binding groupor ZBG (functional group capable of chelating the active site zinc(II)ion), at least one functional group which provides a hydrogen bondinteraction with the enzyme backbone, and one or more side chains whichundergo effective van der Waals interactions with the enzyme subsites.Zinc binding groups in known MMP inhibitors include hydroxamic acids(—C(O)NHOH), reverse hydroxamates (—N(OH)CHO), thiols, carboxylates andphosphonic acids.

[0008] We have discovered a new class of compounds that are inhibitorsof metalloproteinases and are of particular interest in inhibitingMMP13. The compounds of this invention have beneficial potency and/orpharmacokinetic properties. In particular they show selectivity forMMP13.

[0009] MMP13, or collagenase 3, was initially cloned from a cDNA libraryderived from a breast tumour [J. M. P. Freije et al. (1994) Journal ofBiological Chemistry 269(24):16766-16773]. PCR-RNA analysis of RNAs froma wide range of tissues indicated that MMP13 expression was limited tobreast carcinomas as it was not found in breast fibroadenomas, normal orresting mammary gland, placenta, liver, ovary, uterus, prostate orparotid gland or in breast cancer cell lines (T47-D, MCF-7 and ZR75-1).Subsequent to this observation MMP13 has been detected in transformedepidermal keratinocytes [N. Johansson et al., (1997) Cell Growth Differ.8(2):243-250], squamous cell carcinomas [N. Johansson et al., (1997) Am.J. Pathol. 151(2):499-508] and epidermal tumours [K. Airola et al.,(1997) J. Invest. Dermatol. 109(2):225-231]. These results aresuggestive that MMP13 is secreted by transformed epithelial cells andmay be involved in the extracellular matrix degradation and cell-matrixinteraction associated with metastasis especially as observed ininvasive breast cancer lesions and in malignant epithelia growth in skincarcinogenesis.

[0010] Recent published data implies that MMP13 plays a role in theturnover of other connective tissues. For instance, consistent withMMP13's substrate specificity and preference for degrading type IIcollagen [P. G. Mitchell et al., (1996) J. Clin. Invest. 97(3):761-768;V. Knauper et al., (1996) The Biochemical Journal 271:1544-1550], MMP13has been hypothesised to serve a role during primary ossification andskeletal remodelling [M. Stahle-Backdahl et al., (1997) Lab. Invest.76(5):717-728; N. Johansson et al., (1997) Dev. Dyn. 208(3):387-397], indestructive joint diseases such as rheumatoid and osteo-arthritis [D.Wernicke et al., (1996) J. Rheumatol. 23:590-595; P. G. Mitchell et al.,(1996) J. Clin. Invest. 97(3):761-768; O. Lindy et al., (1997) ArthritisRheum 40(8): 1391-1399]; and during the aseptic loosening of hipreplacements [S. Imai et al., (1998) J. Bone Joint Surg. Br.80(4):701-710]. MMP13 has also been implicated in chronic adultperiodontitis as it has been localised to the epithelium of chronicallyinflamed mucosa human gingival tissue [V. J. Uitto et al., (1998) Am. J.Pathol 152(6):1489-1499] and in remodelling of the collagenous matrix inchronic wounds [M. Vaalamo et al., (1997) J. Invest. Dermatol.109(1):96-101].

[0011] U.S. Pat. No. 6,100,266 and WO-99/38843 disclose compounds of thegeneral formula

B-X-(CH₂)_(m)—(CR¹R²)_(n)—W—COY

[0012] for use in the manufacture of a medicament for the treatment orprevention of a condition associated with matrix metalloproteinases.Specifically disclosed is the compoundN-{1S-[4-(4-Chlorophenyl)piperazine-1-sulfonylmethyl]-2-methylpropyl}-N-hydroxyformamide.

[0013] WO-00/12478 discloses arylpiperazines that are matrixmetalloproteinase inhibitors, including compounds with an hydroxamicacid zinc binding group and compounds with a reverse hydroxamate zincbinding group.

[0014] We have now discovered compounds that are potent MMP13 inhibitorsand have desirable activity profiles.

[0015] In a first aspect of the invention we now provide a compound ofthe formula I

[0016] wherein

[0017] B is selected from H, C₁₋₆alkyl, up to C12 cycloalkyl, up to C12aryl, and up to C12 heteroaryl;

[0018] B is optionally substituted by up to three groups independentlyselected from OH, NO₂, CF₃, CN, halogen, SC₁₋₄alkyl, SOC₁₋₄alkyl,SO₂C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₄alkoxy;

[0019] L₁ and L₂ are each independently selected from a direct bond andC₁₋₆alkyl;

[0020] M₁, M₂, M₃, M₄ and M₅ are each independently selected from N andC;

[0021] R1 is the group -X-Y;

[0022] X is C₁₋₆alkyl;

[0023] Y is selected from up to C10 cycloallyl, up to C10 aryl, and upto C10 heteroaryl;

[0024] Y is optionally substituted by up to three groups independentlyselected from OH, NO₂, CF₃, CN, halogen, SC₁₋₄alkyl, SOC₁₋₄alkyl,SO₂C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₄alkoxy;

[0025] Any alkyl group outlined above may be straight chain or branched;

[0026] Any heteroaryl group outlined above is an aromatic ringcontaining one or more heteroatoms independently selected from N, O, S.

[0027] In a further aspect of the invention we provide compounds of theformula II

[0028] wherein B, L₁, L₂, M₁, M₂, M₃, M₄, M₅, R1, X and Y are as definedabove for the compound of formula I.

[0029] Preferred compounds of the formulae I or II are those wherein anyone or more of the following apply:

[0030] B is selected from H, C₁₋₆alkyl, C6 aryl and up to C6 heteroaryl;preferably B is H, C₂₋₄alkyl, C6 aryl, and up to C6 heteroaryl; mostpreferably B is up to C6 heteroaryl;

[0031] B is not substituted or is substituted by at least one groupselected from CF₃, CN, halogen (preferably fluoro or chloro), C₁₋₄alkyl;

[0032] At least one of L₁ and L₂ is a direct bond; preferably each of L₁and L₂ is a direct bond;

[0033] M₁ is N;

[0034] At least one of M₂, M₃, M₄, M₅ is C, and at least one of M₂, M₃,M₄, M₅ is N; preferably each of M₄ and M₅ is C, and at least one of M₂and M₃ is N; most preferably each of M₄ and M₅ is C, M₂ is C or N and M₃is N;

[0035] X is C₂₋₅alkyl; preferably X is C₂₋₃alkyl;

[0036] Y is C6 aryl or C6 heteroaryl; preferably Y is phenyl, pyridyl,pyrimidinyl, or pyrazinyl; most preferably Y is pyrimidinyl;

[0037] Y is not substituted or is substituted by at least one halogengroup (preferably fluoro or chloro).

[0038] For example, preferred compounds of the invention include thosewherein B is phenyl, pyridyl, pyrimidinyl, or thienyl.

[0039] Other preferred compounds include those wherein L₁ is a directbond, L₂ is a direct bond, M₁ is N, M₂ is C or N, M₃ is N, M₄ is C, andM₅ is C.

[0040] Other preferred compounds include those wherein R1 is 3- or4-chlorophenylethyl, 3- or 4-chlorophenylpropyl, 2- or 3-pyridylethyl,2- or 3-pyridylpropyl, 2- or 4-pyrimidinylethyl (optionallymonosubstituted by fluoro or chloro), 2- or 4-pyrimidinylpropyl(optionally monosubstituted by fluoro or chloro), 2-(2-pyrimidinyl)ethyl(optionally monosubstitued by fluoro or chloro), 2-(2-pyrimidinyl)propyl(optionally monosubstitued by fluoro or chloro). Particularly preferredcompounds include those wherein R1 is 2-pyrimidinylpropyl,2-pyrimidinylethyl or 5-fluoro-2-pyrimudinylethyl.

[0041] It will be appreciated that the particular substituents andnumber of substituents on B and/or R1 are selected so as to avoidsterically undesirable combinations.

[0042] Each exemplified compound represents a particular and independentaspect of the invention.

[0043] Where optically active centres exist in the compounds of formulaeI or II, we disclose all individual optically active forms andcombinations of these as individual specific embodiments of theinvention, as well as their corresponding racemates.

[0044] It will be appreciated that the compounds according to theinvention can contain one or more asymmetrically substituted carbonatoms. The presence of one or more of these asymmetric centres (chiralcentres) in a compound of formulae I or II can give rise tostereoisomers, and in each case the invention is to be understood toextend to all such stereoisomers, including enantiomers anddiastereomers, and mixtures including racemic mixtures thereof.

[0045] Where tautomers exist in the compounds of formulae I or II, wedisclose all individual tautomeric forms and combinations of these asindividual specific embodiments of the invention.

[0046] As previously outlined the compounds of the invention aremetalloproteinase inhibitors, in particular they are inhibitors ofMMP13. Each of the above indications for the compounds of the formulae Ior II represents an independent and particular embodiment of theinvention. Whilst we do not wish to be bound by theoreticalconsiderations, the compounds of the invention are believed to showselective inhibition for any one of the above indications relative toany MMP1 inhibitory activity, by way of non-limiting example they mayshow 100-1000 fold selectivity over any MMP1 inhibitory activity.

[0047] The compounds of the invention may be provided aspharmaceutically acceptable salts. These include acid addition saltssuch as hydrochloride, hydrobromide, citrate and maleate salts and saltsformed with phosphoric and sulphuric acid. In another aspect suitablesalts are base salts such as an alkali metal salt for example sodium orpotassium, an alkaline earth metal salt for example calcium ormagnesium, or organic amine salt for example triethylamine.

[0048] They may also be provided as in vivo hydrolysable esters. Theseare pharmaceutically acceptable esters that hydrolyse in the human bodyto produce the parent compound. Such esters can be identified byadministering, for example intravenously to a test animal, the compoundunder test and subsequently examining the test animal's body fluids.Suitable in vivo hydrolysable esters for carboxy include methoxymethyland for hydroxy include formyl and acetyl, especially acetyl.

[0049] In order to use a compound of the formulae I or II or apharmaceutically acceptable salt or in vivo hydrolysable ester thereoffor the therapeutic treatment (including prophylactic treatment) ofmammals including humans, it is normally formulated in accordance withstandard pharmaceutical practice as a pharmaceutical composition.

[0050] Therefore in another aspect the present invention provides apharmaceutical composition which comprises a compound of the formulae Ior II or a pharmaceutically acceptable salt or an in vivo hydrolysableester and pharmaceutically acceptable carrier.

[0051] The pharmaceutical compositions of this invention may beadministered in standard manner for the disease or condition that it isdesired to treat, for example by oral, topical, parenteral, buccal,nasal, vaginal or rectal adminstration or by inhalation. For thesepurposes the compounds of this invention may be formulated by meansknown in the art into the form of, for example, tablets, capsules,aqueous or oily solutions, suspensions, emulsions, creams, ointments,gels, nasal sprays, suppositories, finely divided powders or aerosolsfor inhalation, and for parenteral use (including intravenous,intramuscular or infusion) sterile aqueous or oily solutions orsuspensions or sterile emulsions.

[0052] In addition to the compounds of the present invention thepharmaceutical composition of this invention may also contain, or beco-administered (simultaneously or sequentially) with, one or morepharmacological agents of value in treating one or more diseases orconditions referred to hereinabove.

[0053] The pharmaceutical compositions of this invention will normallybe administered to humans so that, for example, a daily dose of 0.5 to75 mg/kg body weight (and preferably of 0.5 to 30 mg/kg body weight) isreceived. This daily dose may be given in divided doses as necessary,the precise amount of the compound received and the route ofadministration depending on the weight, age and sex of the patient beingtreated and on the particular disease or condition being treatedaccording to principles known in the art.

[0054] Typically unit dosage forms will contain about 1 mg to 500 mg ofa compound of this invention.

[0055] Therefore in a further aspect, the present invention provides acompound of the formulae I or II or a pharmaceutically acceptable saltor in vivo hydrolysable ester thereof for use in a method of therapeutictreatment of the human or animal body. In particular we disclose use inthe treatment of a disease or condition mediated by MMP13.

[0056] In yet a further aspect the present invention provides a methodof treating a metalloproteinase mediated disease or condition whichcomprises administering to a warm-blooded animal a therapeuticallyeffective amount of a compound of the formulae I or II or apharmaceutically acceptable salt or in vivo hydrolysable ester thereof.Metalloproteinase mediated diseases or conditions include arthritis(such as osteoarthritis), atherosclerosis, chronic obstructive pulmonarydiseases (COPD).

[0057] In another aspect the present invention provides a process forpreparing a compound of the formulae I or II or a pharmaceuticallyacceptable salt or in vivo hydrolysable ester thereof which process isoutlined below.

[0058] A compound of the formula I can be prepared from a compound ofthe formula III by reaction with hydroxylamine followed by formylation.

[0059] A compound of the formula III can be prepared from a compound ofthe formula IV and a compound of the formula V or from a compound of theformula VI and a compound of the formula VII. A compound of the formulaV may be prepared from a compound of the formula VII.

[0060] A compound of the formula VII may be prepared from a compound ofthe formula VIII by reaction with a suitable aldehyde (R1CHO) or asuitable ester (R1COOR). A compound of formula VIII may be convienientlyprepared from a compound of formula IX. A compound of the formula IX canbe prepared conveniently from a compound of the formula X and a compoundof the formula XI (where P is hydrogen or a suitable protecting groupand M₁′ is hydrogen or a suitably reactive group).

[0061] A compound of the formula III can also be prepared from acompound of the formula XII by reaction with a suitable aldehyde (R1CHO)or a suitable ester (R1COOR). A compound of the formula XII can beprepared conveniently from a compound of the formula XIII and is acompound of the formula XIV

[0062] A compound of the formula II may be prepared from a compound ofthe formula XV by similar methodology to that described for compound Iabove.

[0063] It will be appreciated that many of the relevant startingmaterials are commercially available or may, be made by any convenientmethod as described in the literature or known to the skilled chemist ordescribed in the Examples herein.

[0064] The compounds of the invention may be evaluated for example inthe following assays:

[0065] Isolated Enzyme Assays

[0066] Matrix Metalloproteinase Family Including for Example MMP13.

[0067] Recombinant human proMMP13 may be expressed and purified asdescribed by Knauper et al., [V. Knauper et al., (1996) The BiochemicalJournal 271:1544-1550 (1996)]. The purified enzyme can be used tomonitor inhibitors of activity as follows: purified proMMP13 isactivated using 1 mM amino phenyl mercuric acid (APMA), 20 hours at 21°C.; the activated MMP13 (11.25 ng per assay) is incubated for 4-5 hoursat 35° C. in assay buffer (0.1M Tris-HCl, pH 7.5 containing 0.1M NaCl,20mM CaCl2, 0.02 mM ZnCl and 0.05% (w/v) Brij 35 using the syntheticsubstrate7-methoxycoumarin-4-yl)acetyl.Pro.Leu.Gly.Leu.N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl.Ala.Arg.NH₂in the presence or absence of inhibitors. Activity is determined bymeasuring the fluorescence at λex 328 nm and λem 393 mn. Percentinhibition is calculated as follows: % Inhibition is equal to the[Fluorescence_(plus inhibitor)−Fluorescence_(background)] divided by the[Fluorescence_(minus inhibitor) 31 Fluorescence_(background)].

[0068] A similar protocol can be used for other expressed and purifiedpro MMPs using substrates and buffers conditions optimal for theparticular MMP, for instance as described in C. Graham Knight et al.,(1992) FEBS Lett. 296(3):263-266.

[0069] Adamalysin Family Including for Example TNF Convertase

[0070] The ability of the compounds to inhibit proTNFα convertase enzymemay be assessed using a partially purified, isolated enzyme assay, theenzyme being obtained from the membranes of THP-1 as described by K. M.Mohler et al., (1994) Nature 370:218-220. The purified enzyme activityand inhibition thereof is determined by incubating the partiallypurified enzyme in the presence or absence of test compounds using thesubstrate 4′,5′-Dimethoxy-fluoresceinylSer.Pro.Leu.Ala.Gln.Ala.Val.Arg.Ser.Ser.Ser.Arg.Cys(4-(3-succinimid-1-yl)-fluorescein)-NH₂in assay buffer (50 mM Tris HCl, pH 7.4 containing 0.1% (w/v) TritonX-100 and 2 mM CaCl₂), at 26° C. for 18 hours. The amount of inhibitionis determined as for MMP13 except λex 490 nm and λem 530 nm were used.The substrate was synthesised as follows. The peptidic part of thesubstrate was assembled on Fmoc-NH-Rink-MBHA-polystyrene resin eithermanually or on an automated peptide synthesiser by standard methodsinvolving the use of Fmoc-amino acids andO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU) as coupling agent with at least a 4- or 5-fold excess ofFmoc-amino acid and HBTU. Ser¹ and Pro² were double-coupled. Thefollowing side chain protection strategy was employed; Ser¹(But),Gln⁵(Trityl), Arg^(8,12)(Pmc or Pbf), Ser^(9,10,11)(Trityl),Cys¹³(Trityl). Following assembly, the N-terminal Fmoc-protecting groupwas removed by treating the Fmoc-peptidyl-resin with 20% piperidine inDMF. The amino-peptidyl-resin so obtained was acylated by treatment for1.5-2 hr at 70° C. with 1.5-2 equivalents of4′,5′-dimethoxy-fluorescein-4(5)-carboxylic acid [Khanna & Ullman,(1980) Anal Biochem. 108:156-161) which had been preactivated withdiisopropylcarbodiimide and 1-hydroxybenzotriazole in DMF]. Thedimethoxyfluoresceinyl-peptide was then simultaneously deprotected andcleaved from the resin by treatment with trifluoroacetic acid containing5% each of water and triethylsilane. The dimethoxyfluoresceinyl-peptidewas isolated by evaporation, trituration with diethyl ether andfiltration. The isolated peptide was reacted with4-(N-maleimido)-fluorescein in DMF containing diisopropylethylamine, theproduct purified by RP-HPLC and finally isolated by freeze-drying fromaqueous acetic acid. The product was characterised by MALDI-TOF MS andamino acid analysis.

[0071] Natural Substrates

[0072] The activity of the compounds of the invention as inhibitors ofaggrecan degradation may be assayed using methods for example based onthe disclosures of E. C. Arner et al., (1998) Osteoarthritis andCartilage 6:214-228; (1999) Journal of Biological Chemistry, 274 (10),6594-6601 and the antibodies described therein. The potency of compoundsto act as inhibitors against collagenases can be determined as describedby T. Cawston and A. Barrett (1979) Anal. Biochem. 99:340-345.

[0073] Inhibition of Metalloproteinase Activity in Cell/Tissue BasedActivity Test as an Agent to Inhibit Membrane Sheddases such as TNFConvertase

[0074] The ability of the compounds of this invention to inhibit thecellular processing of TNFα production may be assessed in THP-1 cellsusing an ELISA to detect released TNF essentially as described K. M.Mohler et al., (1994) Nature 370:218-220. In a similar fashion theprocessing or shedding of other membrane molecules such as thosedescribed in N. M. Hooper et al., (1997) Biochem. J. 321:265-279 may betested using appropriate cell lines and with suitable antibodies todetect the shed protein.

[0075] Test as an Agent to Inhibit Cell Based Invasion

[0076] The ability of the compound of this invention to inhibit themigration of cells in an invasion assay may be determined as describedin A. Albini et al., (1987) Cancer Research 47:3239-3245.

[0077] Test as an Agent to Inhibit Whole Blood TNF Sheddase Activity

[0078] The ability of the compounds of this invention to inhibit TNFαproduction is assessed in a human whole blood assay where LPS is used tostimulate the release of TNFα. Heparinized (10 Units/mL) human bloodobtained from volunteers is diluted 1:5 with medium(RPMI1640+bicarbonate, penicillin, streptomycin and glutamine) andincubated (160 μl) with 20 μl of test compound (triplicates), in DMSO orappropriate vehicle, for 30 min at 37° C. in a humidified (5% CO₂/95%air) incubator, prior to addition of 20 μl LPS (E. coli. 0111:B4; finalconcentration 10 μg/mL). Each assay includes controls of diluted bloodincubated with medium alone (6 wells/plate) or a known TNFα inhibitor asstandard. The plates are then incubated for 6 hours at 37° C.(humidified incubator), centrifuged (2000 rpm for 10 min; 4° C.), plasmaharvested (50-100 μL) and stored in 96 well plates at −70° C. beforesubsequent analysis for TNFα concentration by ELISA.

[0079] Test as an Agent to Inhibit in Vitro Cartilage Degradation

[0080] The ability of the compounds of this invention to inhibit thedegradation of the aggrecan or collagen components of cartilage can beassessed essentially as described by K. M. Bottomley et al., (1997)Biochem J.323:483-488.

[0081] Pharmacodynamic Test

[0082] To evaluate the clearance properties and bioavailability of thecompounds of this invention an ex vivo pharmacodynamic test is employedwhich utilises the synthetic substrate assays above or alternativelyHPLC or Mass spectrometric analysis. This is a generic test which can beused to estimate the clearance rate of compounds across a range ofspecies. Animals (e,g. rats, marmosets) are dosed iv or po with asoluble formulation of compound (such as 20% w/v DMSO, 60% w/v PEG400)and at subsequent time points (e.g. 5, 15, 30, 60, 120, 240, 480, 720,1220 mins) the blood samples are taken from an appropriate vessel into10U heparin. Plasma fractions are obtained following centrifugation andthe plasma proteins precipitated with acetonitrile (80% w/v finalconcentration). After 30 mins at −20° C. the plasma proteins aresedimented by centrifugation and the supernatant fraction is evaporatedto dryness using a Savant speed vac. The sediment is reconstituted inassay buffer and subsequently analysed for compound content using thesynthetic substrate assay. Briefly, a compound concentration-responsecurve is constructed for the compound undergoing evaluation. Serialdilutions of the reconstituted plasma extracts are assessed for activityand the amount of compound present in the original plasma sample iscalculated using the concentration-response curve taking into accountthe total plasma dilution factor.

[0083] In Vivo Assessment

[0084] Test as an Anti-TNF Agent

[0085] The ability of the compounds of this invention as ex vivo TNFαinhibitors is assessed in the rat. Briefly, groups of male WistarAlderley Park (AP) rats (180-210 g) are dosed with compound (6 rats) ordrug vehicle (10 rats) by the appropriate route e.g. peroral (p.o.),intraperitoneal (i.p.), subcutaneous (s.c.). Ninety minutes later ratsare sacrificed using a rising concentration of CO₂ and bled out via theposterior vena cavae into 5 Units of sodium heparin/mL blood. Bloodsamples are immediately placed on ice and centrifuged at 2000 rpm for 10mnin at 4° C. and the harvested plasmas frozen at −20° C. for subsequentassay of their effect on TNFα production by LPS-stimulated human blood.The rat plasma samples are thawed and 175 μl of each sample are added toa set format pattern in a 96 well plate. Fifty pl of hepaeiieed humanblood is then added to each well, mixed and the plate is incubated for30 min at 37° C. (humidified incubator). LPS (25 μl; final concentration10 μg/mL) is added to the wells and incubation continued for a further5.5 hours. Control wells are incubated with 25 μl of medium alone.Plates are then centrifuged for 10 min at 2000 rpm and 200 μl of thesupernatants are transferred to a 96 well plate and frozen at −20° C.for subsequent analysis of TNF concentration by ELISA.

[0086] Data analysis by dedicated software calculates for eachcompound/dose:

Percent inhibition of TNFα=Mean TNFα(Controls)−Mean TNFα(Treated)×100Mean TNFα(Controls)

[0087] Test as an Anti-Arthritic Agent

[0088] Activity of a compound as an anti-arfritic is tested in thecollagen-induced arthritis (CIA) as defined by D. E. Trentham et al.,(1977) J. Exp. Med. 146:857. In this model acid soluble native type IIcollagen causes polyarthritis in rats when administered in Freundsincomplete adjuvant. Similar conditions can be used to induce arthritisin mice and primates.

[0089] Test as an Anti-Cancer Agent

[0090] Activity of a compound as an anti-cancer agent may be assessedessentially as described in I. J. Fidler (1978) Methods in CancerResearch 15:399-439, using for example the B16 cell line (described inB. Hibner et al., Abstract 283 p75 10th NCI-EORTC Symposium, AmsterdamJun. 16-19 1998).

[0091] The invention will now be illustrated but not limited by thefollowing Examples:.

EXAMPLE 1hydroxy{-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyI)methyl]-4-pyrimidin-2-ylbutyl}formamide

[0092]

[0093] To formic acid (1.5 mL, 39 mmol) at 0° C. was added aceticanhydride (375 μL, 3.9 mmol) and the mixture was stirred at RT for 10minutes. The reaction was then recooled to 0° C., and a solution of2-[4-(hydroxyamino)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pentyl]pyrimidine(403 mg, 0.79 mmol) in THF (8 mL) was then added. The reaction wasbrought to RT and stirred for one hour. Volatiles were then removed invacuo, and the residue azeotroped with toluene (2×5 mL). The residue wasthen dissolved in MeOH (10 mL) and heated to 40° C. for one hour. Thesolution was then cooled to RT and concentrated in vacuo. The residuewas then purified by flash chromatography (silica gel, 10% MeOH inEtOAc) to give the title compound as a pale yellow foam (231 mg, 0.43mmol, 54%).

[0094]¹H NMR (DMSO-D6, 373K): 9.41 (br s, 1 H), 8.65 (d, 2H), 8.56 (d,1H), 8.36 (d, 1 H), 8.11 (br s, 1 H), 7.79 (m, 1 H), 7.71 (dd, 1 H),7.55 (d, 1 H), 7.32 (m, 1 H), 7.28 (dd, 1 H), 6.88 (d, 1 H), 3.72 (m, 4H), 3.45 (dd, 1 H), 3.32 (m, 4 H), 3.17 (dd, 1 H), 2.92 (m, 3 H), 1.77(m,4H)

[0095] MS (ESI): 536.45 (MH⁺)

[0096] The starng material was prepared as follows:

[0097] To a stirred solution of 2-chloro-5-iodo-pyridine (CAS number69045-79-0, 10.52 g, 43.9 mmol) and diisopropylethylamine (11.5 mL, 65.9mmol) in DMA (200 mL) was added piperazine (15.14 g, 0.176 mol). Themixture was then heated to 120° C. for 20 hours. The solid precipitatewas then filtered off, and the filtrate evaporated in vacuo. The residuewas partitioned between EtOAc (100 mL) and water (100 mL) and the layerswere separated. The aqueous layer was then extracted with EtOAc (2×100mL). The combined organic extracts were then dried, (MgSO₄), filteredand concentrated in vacuo, to give 1-(5-iodopyridin-2-yl)piperazine as ayellow solid (10.42 g, 36 mmol, 82%).

[0098]¹H NMR (CDCl₃): 8.30 (d, 1 H), 7.65 (dd, 1 H), 6.45 (d, 1 H),3.46, (t, 4 H), 2.97 (t, 4 H).

[0099] MS (ESI): 290.29 (MH⁺)

[0100] To a stirred solution of 1-(5-iodopyridin-2-yl)piperazine (CASnumber 219635-89-9, 10.42 g, 36 mmol), in CH₂Cl₂ (150 mL) at 0° C. wasadded a solution of BOC-O-BOC (7.87 g, 26 mmol). The reaction wasallowed to warm to RT and was stirred for 18 hours. Volatiles wereremoved in vacuo, and the residue was dissolved in EtOAc (200 mL). Thesolution was washed with water (100 mL), and the aqueous layer wasextracted with EtOAc (2×100 mL). The combined organic extracts were thendried, (MgSO₄), filtered and concentrated in vacuo, to give tert-butyl4-(5-iodopyridin-2-yl)piperazine-1-carboxylate as a yellow solid (14 g,36 mmol, 99%).

[0101]¹H NMR (CDCl₃): 8.31 (d, 1 H), 7.08 (dd, 1 H), 6.46 (d, 1 H), 3.50(s, 8H), 1.48 (s, 9 H).

[0102] MS (ESI): 390.37 (MH⁺)

[0103] To a stirred solution of tert-butyl4-(5-iodopyridin-2-yl)piperazine-1-carboxylate (4.087 g, 10.5 mmol) inDMA (75 mL) at RT was added 2-ethynylpyridine, (CAS number 1945-84-2,1.17 mL, 11.5 mmol), triethylanmne (4.4 mL, 31.5 mmol), CuI (800 mg, 4.2mmol) and Pd(PPh₃)₄ (1.21 g, 1.05 mmol). After stirnng for 30 minutes,the DMA was removed in vacuo, and the residue diluted with EtOAc (100mL). The dark solution was then washed with water (100 mL). The layerswere separated and the aqueous phase extracted with EtOAc (3×50 mL). Thecombined organic extracts were then dried, (MgSO₄), filtered andconcentrated in vacuo. The residue was then purified by flashchromatography (silica gel, 50% EtOAc in hexanes) to give tert-butyl4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine-1carboxylate as abrown oil (3.8 g, 10.5 mmol, 99%).

[0104]¹H NMR (CDCl₃): 8.62 (m, 1 H), 8.43 (d, 1 H), 7.68 (m, 2 H), 7.49(d, 1 H), 7.21 (dd, 1 H), 6.58 (d, 1 H), 3.61 (m, 8 H), 1.51 (s, 9 H).

[0105] MS (ESI): 364.94 (MH⁺)

[0106] To a stirred solution of tert-butyl4-[5-(pyridin-2-ylethynyl)pyridin-2-yl)piperazine-1-carboxylate (3.8 g,10.4 mmol) in CH₂Cl₂ (35 mL) at 0° C. was added trifluoroacetic acid(17.2 mL). The reaction was brought to room temperature and stirred for30 minutes. Volatiles were removed in vacuo, and the residue wasazeotroped with toluene (2×10 mL). The residue was then dissolved inCH₂Cl₂ and washed with aqueous sodium hydroxide solution (2M, 2×20 mL).The layers were then separated and the aqueous phase extracted withCH₂Cl₂ (3×10 mL). The combined organic extracts were then dried,(MgSO₄), filtered and concentrated in vacuo. The residue was dissolvedin CH₂Cl₂ (50 mL), and cooled to 0° C. and treated sequentially withtriethylamine (1.5 mL, 16.7 mmol) and methariesulfonyl chloride (1.1 mL,13.5 mmol). The reaction was brought to RT and stirred for 1 hour. Thereaction was then quenched with water (50 mL). The layers were separatedand the aqueous phase extracted with CH₂Cl₂ (3×20 mL). The combinedorganic extracts were then dried, (MgSO₄), filtered and concentrated invacuo. The residue was then purified by flash chromatography (silicagel, 10% MeOH in EtoAc) to give1-(methylsulfonyl)-4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine asa brown solid (3.5 g, 10.2 mmol, 98%).

[0107]¹H NMR (CDCl₃): 8.60 (d, 1 H), 8.46 (d, 1 H), 7.69, (m, 2 H), 7.52(d, 1 H), 7.23 (m, 1 H), 6.65 (d, 1 H), 3.79 (m, 4 H), 3.35 (m, 4 H),2.83 (s, 3 H).

[0108] MS (ESI): 342.83 (MH⁺)

[0109] To a stirred suspension of1-(methylsulfonyl)-4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazine(456 mg, 1.33 mmol) in ThF (10 mL) at −20° C., was added dropwise asolution of LiHMDS in THF (3.0 mL, 1.0M solution, 3.0 mmol). Theresulting suspension was stirred at −20° C. for 30 mninutes before beingtreated with diethyl chlorophosphate (212 μL, 1.46 mmol). The solutionwas then maintained at −20° C. for 15 minutes before being treated witha solution of 4-pyrimidin-2-ylbutanal (220 mg, 1.46 mmol, CAS number260441-10-9) in THF (3 mL). The solution was stirred at −20° C. for afurther 20 minutes before being quenched with saturated aqueous ammoniumchloride solution (10 mL). The layers were separated and the aqueousphase extracted with ethyl acetate (3×10 mL). The combined organicextracts were then dried, (MgSO₄), filtered and concentrated in vacuo togive-[(4E/Z)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pent-4-enyl]pyrimidine.This material was used crude in the next step.

[0110] MS (ESI): 474.97 (MH⁺)

[0111] To a stirred solution of2-[(4E/Z)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pent-4-enyl]pyrimidine(crude from previous step), in THF (15 mL) at RT was added a solution ofhydroxylamine (3 mL, 50% aqueous solution in water). The reaction wasstirred for 3 hours at RT before being quenched with saturated aqueousammonium chloride solution (5 mL). The layers were separated and theaqueous phase extracted with ethyl acetate (3×10 mL). The combinedorganic extracts were then dried, (MgSO₄), filtered and concentrated invacuo. The residue was then purified by flash chromatography (silica,10% MeOH in ethyl acetate) to give2-[4-(hydroxyamino)-5-({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pentyl]pyrimidine(403 mg, 0.79 mmol).

[0112] MS (ESI): 508.22 (MH⁺)

EXAMPLE 2hydroxy[4-pyrimidin-2-yl-1-({[4-(5-15-(trifluoromethyl)pyridin-2-yl]ethynylpyridin-2-yl)piperazin-1-yl]sulfonyl}methyl)butyl]formamide

[0113]

[0114] To formic acid (0.36 mL, 9.5 mmol) at 0° C. was added aceticanhydride (90 μL, 0.95 mmol) and the mixture was stirred at RT for 10minutes. The reaction was then recooled to 0° C., and added to asolution of2-(4-(hydroxyanino)-5-{[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazin1-yl]sulfonyl}pentyl)pyrimidine (403 mg, 0.79 mmol) and formic acid(0.36 mL, 9.5 mmol) in THF (3 mL) at 0° C. The reaction was brought toRT and stirred for one hour. Volatiles were then removed in vacuo, andthe residue azeotroped with toluene (2×5 mL). The residue was thendissolved in MeOH (10 mL) and heated to 40° C. for one hour. Thesolution was then cooled to RT and concentrated in vacuo. The residuewas then purified by flash chromatography (silica gel, 10% MeOH inEtOAc) to give the title compound as a pale yellow foam (66 mg, 0.11mmol, 58%).

[0115]¹H NMR (DMSO-D6, 373K): 9.38 (br s, 1 H), 8.90 (d, 1 H), 8.67 (d,2 H), 8.42 (d, 1 H), 8.15 (dd, 1 H), 8.14 (br s, 1 H), 7.73 (m, 2 H),7.27 (t, 1 H), 6.90 (d, 1 H), 3.71 (mn, 4 H), 3.42 (dd, 1 H), 3.30 (m, 4H), 3.16 (dd, 1 H), 2.88 (m, 3 H), 1.76 (m, 3 H), 1.66 (m, 1H)

[0116] MS (ESI): 604.39 (MH⁺)

[0117] The starting material was prepared as follows:

[0118] To a stirred solution of tert-butyl4-(5-iodopyridin-2-yl)piperazine-1-carboxylate (1.0 g, 2.57 mmol,prepared as in Example 1), trimethylsilyl acetylene (0.73 mL, 5.14mmol), triethylarnine (1.11 mL, 7.71 mmol), and cuprous iodide (196 mg,1.03 mmol) in DMA (20 mL) at 25° C. was added tetrakistriphenylphosphinepalladium (0) (149 mg, 5mol %). The reaction was stirred for 10 minutes.Volatiles were removed in vacuo. The residue was then dissolved in ethylacetate (250 mL) and washed with water (100 mL). The layers were thenseparated and the aqueous phase extracted with ethyl acetate (250 mL).The combined organic extracts were then dried, (MgSO₄), filtered andconcentrated in vacuo. The residue was then purified by flashchromatography (silica gel, 10% EtOAc in hexanes) to give tert-butyl4-{5-[(trimethylsilyl)ethynyl]pyridin-2-yl}piperazine-1-carboxylate as apale brown solid (652 mg, 1.81 mmol, 70%).

[0119]¹H NMR (CDCl₃): 8.28 (d, 1 H), 7.50 (dd, 1 H), 6.50 (d, 1 H), 3.57(m, 8 H), 1.49 (s, 9 H), 0.25 (s, 9H).

[0120] MS (ESI): 360.51 (MH⁺)

[0121] To a stirred solution of tert-butyl4-{5-[(trimethylsilyl)ethynyl]pyridin-2-yl}piperazine-1-carboxylate (637mg, 1.77 mmol), in THF (8 mL) at 25° C. was added tetrabutylammoniumfluoride (1.77 mL, 1.0M in THF). The reaction was stirred for 1 hour.Water (5 mL) was then added and product extracted with ethyl acetate(2×5 mL). The combined organic extracts were then dried, (MgSO₄),filtered and concentrated in vacuo to give tert-butyl4-(5-ethynylpyridin-2-yl)piperazine-1-carboxylate as an orange solid(509 mg, 1.77 mmol, 100%).

[0122]¹H NMR (CDCl₃): 8.30 (d, 1 H), 7.54 (dd, 1 H), 6.52 (d, 1 H), 3.56(m, 8H), 3.07 (s, 1 H), 1.49 (s, 9H).

[0123] To a stirred solution of tert-butyl4-(5-ethynylpyridin-2-yl)piperazine-1-carboxylate (509 mg, 1.77 mmol),2-Bromo-5-trifluoromethyl pyridine (400mg, 1.77 mmol), triethylamine(0.74 mL, 5.31 mmol), and cuprous iodide (135 mg, 0.71 mmol) in DMA (20mL) at 25° C. was added tetrakistriphenylphosphine palladium (0) ( 102mg, 5 mol %). The reaction was then stirred for 1 hour. Volatiles wereremoved in vacuo, and the residue was azeotroped with toluene (2×10 mL).The residue was then dissolved in CH₂Cl₂ (10 mL) and washed with water(10 mL). The layers were then separated and the aqueous phase extractedwith CH₂Cl₂ (2×100 mL). The combined organic extracts were then dried,(MgSO₄), filtered and concentrated in vacuo. The residue was thenpurified by flash chromatography (silica gel, 25% EtOAc in hexanes) togive tert-butyl4-(5-{[5-(tdfluorometyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazine-1-carboxylateas a yellowsolid (729 mg, 1.68 mmol, 95%).

[0124]¹H NMR (CDCl₃): 8.83 (d, 1 H), 8.42 (d, 1 H), 7.88, (dd, 1 I),7.67 (dd, 1 H), 7.56 (d, 1 H), 6.59 (d, 1 H), 3.60 (m, 4 H), 3.53 (m, 4H), 1.50 (s, 9H).

[0125] MS (ESI): 433.49 (MH⁺)

[0126] To a stirred solution of tert-butyl4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazine-1-carboxylate(724 mg, 1.67 mmol) in CH₂Cl₂ (8 mL) at 0° C. was added trifluoroaceticacid (4 mL). The reaction was brought to room temperature and stirredfor 30 minutes. Volatiles were removed in vacuo. The residue was thendissolved in CH₂Cl₂ (20 mL) and washed with aqueous sodium hydroxidesolution (2M, 2×20 mL). The layers were then separated and the aqueousphase extracted with CH₂Cl₂ (3×10 mL). The combined organic extractswere then dried, (MgSO₄), filtered and concentrated in vacuo. Theresidue was dissolved in CH₂Cl₂ (50 mL), and cooled to 0° C. and treatedsequentially with triethylamine (0.25 mL, 1.75 mmol) and methanesulfonylchloride (0.14 mL, 13.5 mmol). The reaction was brought to RT andstirred for 1 hour. The reaction was then quenched with water (50 mL).The layers were separated and the aqueous phase extracted with CH₂Cl₂(3×20 mL). The combined organic extracts were then dried, (MgSO₄),filtered and concentrated in vacuo to give1-(methylsulfonyl)-4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazineas an orange solid (445 mg, 1.08 mmol, 65%).

[0127]¹H NMR (CDCl₃): 8.85 (d, 1 H), 8.45 (d, 1 H), 7.90, (dd, 1 H),7.70 (dd, 1 H), 7.60 (d, 1 H), 6.65 (d, 1 H), 3.79 (m, 4 H), 3.35 (t, 4H), 2.80 (s, 3 H).

[0128] MS (ESI): 411.12 (MH⁺)

[0129] To a stirred suspension of1-(methylsulfonyl)4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazine(437 mg, 1.06 mmol) in THF (10 mL) at −20° C., was added dropwise asolution of LiHMDS in THF (2.34 mL, 1.0M solution, 2.34 mmol). Theresulting suspension was stirred at −20° C. for 30 minutes before beingtreated with diethyl chlorophosphate (0.17 mL, 1.17 mmol). The solutionwas then maintained at −20° C. for 15 minutes before being treated witha solution of 4-pyrimidin-2-ylbutanal (159 mg, 1.06 mmol, CAS number260441-10-9) in THF (3 mL). The solution was stirred at −20° C. for afurther 20 minutes before being quenched with saturated aqueous ammoniumchloride solution (10 mL). The layers were separated and the aqueousphase extracted with ethyl acetate (3×10 mL). The combined organicextracts were then dried, (MgSO₄), filtered and concentrated in vacuo togive2-((4E/Z)-5-{[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrimidine.This was used crude in the next step.

[0130] MS (ESI): 543.19 (MH⁺)

[0131] To a stirred solution of2-((4E/Z)-5-{[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrimidine(235 mg, 0.43 mmol), in THF (5 mL) at RT was added a solution ofhydroxylamine (0.5 mL, 50% aqueous solution in water). The reaction wasstirred for 3 hours at RT before being quenched with saturated aqueousammonium chloride solution (5 mL).The layers were separated and theaqueous phase extracted with ethyl acetate (3×10 mL). The combinedorganic extracts were then dried, (MgSO₄), filtered and concentrated invacuq. The residue was then purified by flash chromatography (silica,10% MeOH in ethyl acetate) to give2-(4-(hydroxyamino)-5-{[4(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazin-1-yl]sulfonyl}pentyl)pyriinidine(126 mg, 51 mmol).

[0132] MS (ESI): 576.35 (MH⁺)

EXAMPLE 3hydroxy{(3S)-3-phenyl-1-[({4-[4-(phenylethynyl)phenyl]piperazin-1-yl}sulfonyl)methyl]butyl}formamide

[0133]

[0134] To formic acid (2.5 mL, 65 mmol) at 0° C. was added aceticanhydride (500 μL, 5.2 mmol) and the mixre was stirred at 0° C. for 10minutes. This was added to a solution of1-{[(4S)-2-(hydroxyamino)-4-phenylpentyl]sulfonyl}-4-[4-(phenylethynyl)phenyl]piperazine(345 mg, 0.685 mmol) in CH₂Cl₂ (5 mL) cooled to 0 ° C. The reaction wasbrought to RT and stirred for one hour. Volatiles were then removed invacuo. The residue was then precipitated in MeOH (10 mL) and heated to40° C. for two hours, dissolving. The solution was then cooled to RT andconcentrated in vacuo. The residue was then purified by flashchromatography (silica gel, 1-2% MeOH in CH₂Cl₂) to give the titlecompound as a white foam (330 mg, 0.621 mmol, 91%).

[0135]¹H NMR (CDCl₃): complex mixture of 4 rotameric diastereomers.

[0136] MS (ESI): 532(MH⁺)

[0137] The starting material was prepared as follows:

[0138] To a solution of 1-(4-bromophenyl)piperazine hydrochloride (5.09g, 18.3 mmol, CAS number 68104-62-1) and triethylamine (7.67 mL), inCH₂Cl₂ was added methanesulfonyl chloride (2.83 mL, 36.3 mmol) dropwise.The mixture was stirred for one hour at RT, then CH₂Cl₂ (100 mL) wasadded. The organics were washed with water (2×100 mL), and evaporated invacuo to give a yellow solid which crystallised from ethanol and waswashed with ether to give 1-(methylsulfonyl)-4-(4-bromophenyl)piperazineas a white powder (4.74 g, 81%).

[0139]¹H NMR (CDCl₃): 7.38 (d, 2 H), 6.91 (d, 2 H), 3.21 (m, 8 H), 2.89(s, 3 H).

[0140] MS (ESI): 318, 320 (MH⁺ Br isotope pattern).

[0141] To a stirred suspension of1-(methylsulfonyl)-4-(4-bromophenyl)piperazine (5.77 g, 18.1 mmol),phenyl acetylene (3.97 mL, 36.1 mmol), Copper (I) iodide (38 mg, 0.2mmol) and triphenylphosphine (95 mg, 0.36 mmol) in piperidine (72 mL) at20° C. was added dichlorobis(triphenylphosphine)palladium(II) (38 mg, 54μmol). The reaction was warmed to 105° C. and stirred for 14 hours underan inert atmosphere. Volatiles were removed in vacuo, and the solidresidue was dissolved in CH₂Cl₂ (200 mL) and washed with aqueoushydrochloric acid (2M) to pH 2. The layers were then separated and theorganic extract dried, (sat Brine and MgSO₄), filtered and evaporated todryness in vacuo. The residue was triturated in CH₂Cl₂, and filtered togive 1-(methylsulfonyl)-4-[4-(phenylethynyl)phenyl]piperazine as a flakywhite solid (1.99 g, 5.86 mmol, 32%).

[0142]¹H NMR (CDCl₃): 7.5 (dd, 2 H), 7.45 (d, 2 H), 7.33, (m,3 H), 6.86(d, 2 H); 3.38 (d, 8 H), 2.82 (s, 3 H).

[0143] MS (ESI): 341 (MH⁺)

[0144] To a stirred suspension of1-(methylsulfonyl)-4-[4-(phenylethynyl)phenyl]piperazine (511 mg, 1.5mmol) in THF (10 mL) at −40° C., was added dropwise a solution of LiHMDSin THF (3.3 mL, 1.0M solution, 3.3 mmol). The resulting suspension wasstirred at −40° C. for 10 minutes before being treated withchlorotrirethylsilane (189 μL, 1.5 mmol). The solution was thenmaintained at −40° C. for 10 minutes before being treated with asolution of (3S)-3-phenylbutanal (267 mg, 1.8 mmol, CAS-number53531-194) in THF (6 mL). The solution was allowed to warm to −15° C.over 1 hour before being quenched with a solution of hydroxylamine (3mL, 50% aqueous solution in water). The reaction was stirred for 3 hoursat RT. The reaction was partitioned between ethyl acetate (30 mL) andwater (30 mL). The combined organic extracts were then dried (sat. brineand MgSO₄), filtered and concentrated in vacuo. The residue was thenpurified by flash chromatography (silica, 35-55% ethyl acetate iniHexane) to give1-{[(4S)-2-(hydroxyamino)4-phenylpentyl]sulfonyl}-4-[4-(phenylethynyl)phenyl]piperazine(351 mg, 0.697 mmol).

[0145]¹H NMR (CDCl₃): diastereomeric: 7.48 (m, 4 H), 7.25 (m, 8H+CHCl₃), 6.87 (q, 2 H), 3.35 (t, 4 H), 3.23 (m, 5.5 H), 2.92 (m, 1 H),2.76 (m, 0.5 H), 2.02 (m, 1 H), 1.83 (m, 1 H), 1.33 (d,3 H).

[0146] MS (ESI): 504 (MH⁺)

EXAMPLE 4hydroxy[(3S)-3-phenyl-1-({[4-(4-ethynylphenyl)piperazin-1-yl]sulfonyl}methyl)butyl]formamide

[0147]

[0148] To formic acid (3.75 mL, 98 mmol) at 0° C. was added aceticanhydride (750 μL, 7.8 mmol) and the mixture was stirred at 0° C. for 10minutes. This was added to a solution of1-{[(4S)-2-(hydroxyamino)-4-phenylpentyl]sulfonyl}-4-[4-(trimethylsilylethynyl)phenyl]piperazine(720 mg, 94 wt %, 1.35 mmol) in CH₂Cl₂ (5 mL) ia cooled to 0° C. Thereaction was brought quickly to RT and stirred for 20 minutes. Volatileswere then removed in vacuo, and the residue azeotroped with toluene (3×5mL). The residue was then dissolved in MeOH (10 mL) and heated to 40° C.for two hours. The solution was then cooled to RT and concentrated invacuo. The residue was then purified by flash chromatography (silicagel, 40-85% EtOAc in iHexane) and evaporated in vacuo. The solid wasdissolved in ether/CH₂Cl₂ and the insoluble contaminant removed byfiltration to give the title compound as a pink foam (90 mg, 0.171 mmol,15%).

[0149]¹H NMR (CDCl₃): complex mixture of 4 rotameric diastereomers.

[0150] MS (ESI): 456 (MH⁺)

[0151] The starting material was prepared as follows:

[0152] To a stirred suspension of1-(methylsulfonyl)-4-(4-bromophenyl)piperazine (4.79 g, 15 mmol,prepared as in example 3), trimethylsilylacetylene (4.15 mL, 30 mmol),Copper (I) iodide (31 mg, 0.17 mmol) and triphenylphosphine (80 mg, 0.3mmol) in piperidine (60 mL) at 20° C. was addeddichlorobis(triphenylphosphine)palladium(II) (31 mg, 45 μmol).

[0153] The reaction was warmed to 105° C. and stirred for 8 hours underan inert atmosphere. After cooling, the suspension was filtered andwashed with toluene. The solid was dissolved in CH₂Cl₂, washed withwater and filtered through silica, washing with CH₂Cl₂. The filtrate wasconcentrated and the resulting suspension filtered, washing well withCH₂Cl₂/iHexane to give1-(methylsulfonyl)-4-[4-(trimethylsilylethynyl)phenyl]piperazine as awhite powder (3.05 g, 9.06 mmol, 60%).

[0154]¹H NMR (CDCl₃): 7.3 (d, 2 H), 6.9 (d, 2 H), 3.33 (t, 4 H), 3.22(t, 4 H), 2.9 (s, 3 H), 0.2 (s, 9H).

[0155] MS (ESI): 337 (MH⁺)

[0156] To a stirred suspension of1-(methylsulfonyl)-4-[4-(trimethylsilylethynyl)phenyl]piperazine (673mg, 2 mmol) in THF (13 mL) at −40° C., was added dropwise a solution ofLiHMDS in THF (4.5 mL, 1.0M solution, 4.5 mmol). The resultingsuspension was stirred at −40° C. for 10 minutes before being treatedwith chlorotrimethylsilane (254 μL, 2 mmol). The solution was thenmaintained at −40° C. for 10 minutes before being treated with asolution of (3S)-3-phenylbutanal (385 mg, 2.6 mmol, CAS number53531-19-4) in ThB (8 mL). The solutionwas allowed to warm to −15° C.over 1 hour before being quenched with a solution of hydroxylamne (1.23mL, 50% aqueous solution in water, 20 mmol). The reaction was stirredfor 3 hours at RT. The reaction was partitioned between ethyl acetate(30 mL) and water (30 mL). The combined organic extracts were thendried, (sat. brine and MgSO₄), filtered and concentrated in vacuo. Theresidue was then purified by flash chromatography (silica, 30-40% ethylacetate in iHexane) to give1-{[(4S)-2-(hydroxyammno)-4-phenylpentyl]sulfonyl}-4-[4-(trimethylsilylethynyl)phenyl]piperazine(720 mg, 1.44 mmol, 68% @ 94 wt %).

[0157]¹H NMR (CDCl₃): diastereomeric: 7.39 (d, 2 H), 7.27 (m, 5H+CHCl₃), 6.82 (q, 2 H), 3.37 (b, 4.5 H), 3.25 (b, 3 H), 3.18 (b,,2 H),2.92 (m, 1 H), 2.75 (m, 0.5 H), 2.02 (m, 1 H), 1.83 (m, 1 H), 1.33 (d, 3H), 0.26 (s, 9H).

[0158] MS (ESI): 500 (MH⁺)

EXAMPLE 5

[0159] The following compounds were also prepared.

Prepared using method in B R1 M + H example H 2-PyrimidinylCH2CH2CH2531.6 2 2-Pyridyl 2-PyrimidinylCH2CH2CH2 536.4 1 tert-butyl2-PyrimidinylCH2CH2CH2 515.5 1 n-propyl 2-PyrimidinylCH2CH2CH2 501.5 12-Pyridyl 5-F-2-PyrimidinylCH2CH2 539.8 1 5-(CF3)-2-Py-2-PyrimidinylCH2CH2CH2 604.4 2 ridyl 2-Pyridyl 2-PyrimidinylCH2CH2 522.11 3-Pyridyl 2-PyrimidinylCH2CH2CH2 536.3 1 4-Pyridyl2-PyrimidinylCH2CH2CH2 536.3 1 2-Pyrimidinyl 2-PyrimidinylCH2CH2CH2537.5 7 2-Pyridyl 5-Cl-2-PyrimidinylCH2CH2 556.21 1

EXAMPLE 6hydroxy{4-pyrimidin-2-yl-1-[({4-[5-(thien-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)methyl]butyl}formamide

[0160]

[0161] To formic acid (0.3 mL, 8 mmol) at 0° C. was added aceticanhydride (75 μL, 0.8 mmol) and the mixture was stirred at RT for 10minutes. The reaction was then recooled to 0° C., and a solution of2-[4-(hydroxyamino)-5-({4-[5-(thien-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pentyl]pyrimidine(84 mg, 0.16 mmol) in THF (8 mL) was then added. The reaction wasbrought to RT and stirred for one hour. Volatiles were then removed invacuo, and the residue azeotroped with toluene (2×5 mL). The residue wasthen dissolved in MeOH (10 mL) and heated to 40° C. for one hour. Thesolution was then cooled to RT and concentrated in vacuo. The residuewas then purified by flash chromatography (silica gel, 10% MeOH inEtOAc) to give the title compound as an off-white foam (87 mg, 0.16mmol, 54%).

[0162]¹H NMR (DMSO-D6, 373K): 9.38 (br s, 1 H), 8.68 (d, 2H), 8.30 (d, 1H), 8.12 (br s, 1 H), 7.64 (dd, 1 H), 7;55 (d, 1 H), 7.28 (dd, 1 H),7.22 (t, 1H), 7.09 (t, 1H), 6.85 (d, 1 H), 3.68 (m, 4 H),;3.41 (dd, 1H), 3.23 (m, 4 H), 3.12 (dd, 1 H), 2.85 (m, 3 H), 1.70 (m, 4 H)

[0163] MS (ESI): 541.46(MH⁺)

[0164] The starting material was prepared as follows:

[0165] To a stirred solution of 2-chloro-5-iodo-pyridine (CAS number69045-79-0, 10.52 g, 43.9 mmol) and diisopropylethylamine (11.5 mL, 65.9mmol) in DMA (200 mL) was added piperazine (15.14 g, 0.176 mol). Themixture was the heated to 120° C. for 20 hours. The solid precipitatewas then filtered off, and the filtrate evaporated in vacuo. The residuewas partitioned between EtOAc (100 mnL) and water (100 mL) and thelayers were separated. The aqueous layer was then extracted with EtOAc(2×100 mL). The combined organic extracts were then dried, (MgSO₄),filtered and concentrated in vacuo, to give1-(5-iodopyridin-2-yl)piperazine as a yellow solid (10.42 g, 36 mmol,82%).

[0166]¹H NMR (CDCl₃): 8.30 (d, 1 H), 7.65 (dd, 1 H), 6.45 (d, 1 H),3.46, (t, 4 H), 2.97 (t, 4 H).

[0167] MS (ESI): 290.29 (MH⁺)

[0168] To a stirred solution of 1-(5-iodopyridin-2-yl)piperazine (CASnumber 219635-89-9, 5.083 g, 17.6 mmol), and triethylamine (4.91 mL,35.2 mmol) in CH₂Cl₂ (75 mL) at 0° C. was added methane sulfonylchloride (2.04 mL, 26.4 mmol) dropwise and the resulting suspensionstirred for 30 mins at room temperature. The reaction mixture wasdiluted with CH₂Cl₂ (200 mL) and washed twice with water (2×200 mL). Theorganic phase was washed with saturated brine solution (1×200 mL), dried(MgSO₄), filtered and concentrated in vacuo to give a dark yellow solid.This was triturated with ether, filtered and dried to give1-(5-iodopyridin-2-yl)-4-(methylsulfonyl)piperazine as a yellow solid(5.378 g, 83%).

[0169]¹H NMR (CDCl₃): 8.33 (d, 1 H), 7.70 (dd, 1 H), 6.48 (d, 1 H),3.63, (t, 4 H), 3.30 (t, 4 H), 2.80 (s, 3 H).

[0170] MS (ESI): 367.99 (MH⁺)

[0171] To a stirred suspension of1-(5-iodopyridin-2-yl)-4-(methylsulfonyl)piperazine (7.760 g, 21.1 mmol)in THF (150 mL) at −20° C., was added dropwise a solution of LiHMDS inTHF (46.4 mL, 1.0M solution, 46.4 mmol). The resulting suspension wasstirred at −20° C. for 30 minutes before being treated with diethylchlorophosphate (3.35 mL, 23.2 mmol). The solution was then maintainedat −20° C. for 15 minutes before being treated with a solution of4-pyrimidin-2-ylbutanal (3.48 g, 23.2 mmol, CAS number 260441-10-9) inTHF (20 mL). The solution was stirred at −20° C. for a further 20minutes before being quenched with saturated aqueous ammonium chloridesolution (100 mL). The layers were separated and the aqueous phaseextracted with ethyl acetate (3×500 mL). The combined organic extractswere then dried, (MgSO₄), filtered and concentrated in vacuo. This waspurified by bond-elut chromatography eluting with 25% EtOAc/hex to 50%EtOAc/hex to 100% EtOAc to give2-((4E/Z)-5-{[4-(5-iodopyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrimidine.

[0172] MS (ESI): 500.20 (MH⁺)

[0173] To a stirred solution of2-((4E/Z)-5-{[4-(5-iodopyridin-2-yl)piperazin-1-ylsulfonyl}pent4-enyl)pyrimidine(1.906 g, 3.82 mmol) in DMA (35 mL) at RT was added trimethylsilylacetylene, (CAS number 1066-54-2, 1.08 mL, 7.64 mmol), triethylamine(1.60 mL, 11.5 mmol), CuI (291 mg, 1.53 mmol) and Pd(PPh₃)₄ (442 mg,0.38 mmol). After stirring for 30 minutes, the DMA was removed in vacuo,and the residue diluted with EtOAc (100 mL). The dark solution was thenwashed with water (100 mL). The layers were separated and the aqueousphase extracted with EtOAc (3×50 mL). The combined organic extracts werethen dried, (MgSO₄), filtered and concentrated in vacuo. The residue wasthen purified by flash chromatography (silica gel, 50% EtOAc in hexanesto 100% EtOAc) to give2-{(4E/Z)-5-[(4-{5-[(trimethylsilyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]pent-4-enyl}pyrimidineas a yellow foam (1.609 g, 3.43 mmol, 90%). This material was usedsemi-crude in the next step.

[0174] To a stirred solution of2-{(4E/Z-5-[(4-{5-[(trimethylsilyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]pentenyl}pyrimidine(1.605 g, 3.42 mmol) in TIF (50 mL) was added TBAF (3.42 mL, 1 Msolution, 3.42 mmol) and stirred at room temperature for 30 mins. Thereaction mixture was then partitioned between water (200 mL) and EtOAc(200 mL).The layers were separated and the aqueous phase extracted twicewith EtOAc (2×100 mL). The combined organics were then washed with brine(200 mL) dried, (MgSO₄), filtered and concentrated in vacuo. The residuewas purified by bond-elut chromatography eluting with 25% EtOAc/hex to50% EtOAc/hex to 100% EtOAc to give2-((4E/Z)-5-{[4-(5-ethynylpyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrimidineas a pale orange solid (1.065 g, 2.68 mmol, 78%).

[0175] MS (ES]): 398.10

[0176] To a stirred solution of2-((4E/)-5-{[4-(5-ethynylpyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrimidine(487 mg, 1.23 mmol) in DMA (20 mL) at RT was added 2-iodothiophene, (CASnumber 516-12-1, 0.17 mL, 1.48 mmol), triethylamine (0.52 mL, 3.69mmol), CuI (94 mg, 0.49 mmol) and Pd(PPh₃)₄ (142 mg, 0.13 mmol). Afterstirring for 1 hour, the DMA was removed in vacuo, and the residuediluted with EtOAc (100 mL). The dark solutibn was then washed withwater (100 mL). The layers were separated and the aqueous phaseextracted with EtOAc (3×50 mL). The combined organic extracts were thendried, (MgSO₄), filtered and concentrated in vacuo. The residue was thenpurified by flash chromatography (silica gel, 50% EtOAc in hexanes toEtOAc) to give2-[(4E/Z)-5-({4-[5-(thien-2-ylethynylpyridin-2-yl]piperazin-1-yl}sulfonyl)pent-4-enyl]pyrimidineas a pale brown oil (371 mg, 0.69 mmol, 56%). This material was usedsemi-crude in the next step.

[0177] To a stirred solution of2-[(4E/Z)-5-({4-[5-(thien-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pent-4-enyl]pyrimidine(371 mg, 0.69 mmol) in THF (4 mL) was added hydroxylamine (0.4 mL, 50%aqueous solution in water) and stirred for 24 hours at roomteimperature. The reaction was partitioned between ethyl acetate (30 mL)and water (30 mL). The combined organic extracts were then dried(MgSO₄), filtered and concentrated in vacuo. The residue was thenpurified by bond elut chromatography (silica gel, EtOAc to 10%MeOH/EtOAc) to give2-[4ydroxyamino)-5-({4-[5-(thien-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)pentyl]pyrimidine(84 mg, 0.16 mmol, 23%). Also recovered some isomerised SM (220 mg, 0.46mmol, 66%) presumably formed from TBAF deprotection of2-((4E/Z)-5-{[4-(5-ethynylpyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrrimie.

[0178]¹H NMR (CDCl₃): 8.66 (m, 2 H), 8.32 (d, 1 H), 7.62 (dd, 1 H), 7.28(m, 2 H), 7.17 (dd, 1 H), 7.02 (m, 1 H), 6.63 (m, 1 H), 5.80 (br s, 1H), 3.74 (m, 4 H), 3.46 (m, 2 H), 3.41 (m, 4 H), 3.19 (m, 1 H), 3.04 (m,1 H), 2.81 (m, 1 H), 2.00 (m, 2 H), 1.69 (m, 2 H).

[0179] MS (ESI): 513.41 (MH⁺)

EXAMPLE 71-{[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]methyl}-4-pyrimidin-2-ylbutyl(hydroxy)formamide

[0180]

[0181] To formic acid (1.85 mL, 49.0 mmol) at 0° C. was added aceticanhydride (0.46 mL, 4.9 mmol) and the mixture was stirred at RT for 10minutes. The reaction was then recooled to 0° C., and a solution of2-[5-[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]-4-(hydroxyamino)pentyl]pyriridine(515 mg, 0.98 mmol) in THF (10 mL) was then added. The reaction wasbrought to RT and stirred for one hour. Volatiles were then removed invacuo, and the residue azeotroped with toluene (2×5 mL). The residue wasthen dissolved in MeOH (10 mL) and heated to 40° C. for one hour. Thesolution was then cooled to RT and concentrated in vacuo. The whitesolid which precipitates upon cooling to room temperature was filteredoff and dried (258 mg, 0.47 mmol, 48%).

[0182]¹H NMR (DMSO-D6, 373K): 9.40 (br s, 1 H), 8.67 (d, 2H), 8.31 (d, 1H), 8.12 (br s, 1 H), 7.68 (dd, 1 H), 7.55 (m, 2 H), 7.24 (t, 1 H), 7.19(t, 2 H), 6.87 (d, 1 H), 4.34 (br s, 1 H), 3.67 (t, 4 H), 3.45 (dd, 1H), 3.29 (t, 4 H), 3.13 (dd, 1 H), 2.89 (t 2 H), 1.72 (m, 4 H)

[0183] MS (ESI): 553.54 (MH⁺)

[0184] The starting material was prepared as follows:

[0185] To a stirred solution of((4E/Z)-5-{[4-(5-iodopyridin-2-yl)piperazin-1-yl]sulfonyl}pent-4-enyl)pyrimidine(988 mg, 1.98 mmol, prepared as in example 6) in DMA (25 mL) at RT wasadded 4-ethynylfluorobenzene, (CAS number 766-98-3, 476 mg, 3.96 mmol),triethylamine (0.83 mL, 5.94 mmol), CuI (151 mg, 0.8 mmol) and Pd(PPh₃)₄(229 mg, 0.2 mmol). After stirring for 60 minutes, the DMA was removedin vacuo, and the residue diluted with EtOAc (100 mL). The dark solutionwas then washed with water (100 mL). The layers were separated and theaqueous phase extracted with EtOAc (3×50 mL). The combined organicextracts were then dried, (MgSO₄), filtered and concentrated in vacuo:The residue was then purified by bond elut chromatography (silica gel,50% EtOAc in hexanes to 100% EtOAc) to give2-{(4E/)-5-[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]pent-4-enyl}pyrimidineas a pale brown solid (765 g, 1.56 mmol, 79%). This was used semi-crudein the next step.

[0186] To a solution of2-{(4EZ)-5-[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]pent-4-enyl}pyrimidine(760 mg, 1.55 mmol) in THF (10 mL) was added a solution of hydroxylamine(2 mL, 50% aqueous solution in water). The reaction was stirred for 2hours at RT before being quenched with saturated aqueous ammoniumchloride solution (5 mL). The layers were separated and the aqueousphase extracted with ethyl acetate (3×20 mL). The combined organicextracts were then dried, (NgSO₄), filtered and concentrated in vacuo.The residue was then purified by bond elut (silica, EtOAc to 10% MeOH inethyl acetate) to give2-[5-[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]4-(hydroxyamino)pentyl]pyrimidine(518 mg, 0.99 mmol, 64%).

[0187] MS (ESI): 525.33 (MH⁺)

EXAMPLE 8hydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)methyl]4-pyrimidin-2-ylbutyl}formamide

[0188]

[0189] The racernic mixture, prepared as in example 1, was separated bychiral HPLC (on a Daicel Chiralpak AD column, 1 micron, 2 cm×25 cm, 20%MeOH/MeCN eluent, flow rate 9 mL/min) to givehydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)methyl]-4-pyrimidin-2-ylbutyl}formamideas a white solid.

[0190]¹H NMR (DMSO-D6, 373K): 9.41 (br s, 1 H), 8.65 (d, 2H), 8.56 (d,1H), 8.36 (d, 1 H), 8.11 (br s, 1 H), 7.79 (m, 1 H), 7.71 (dd, 1 H),7.55 (d, 1 H), 7.32 (m, 1 H), 7.28 (dd, 1 H), 6.88 (d, 1 H), 3.72 (m, 4H), 3.45 (dd, 1 H), 3.32 (m, 4 H), 3.17 (dd, 1 H), 2.92 (m, 3 H),1.77(m,4H)

[0191] MS (ESI): 536.45 (MH⁺)

EXAMPLE 9hydroxy{1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl}sulfonyl)methyl]-4-pyrimidin-2-ylbutyl}formamide

[0192]

[0193] Formic acid (1.8 mL, 50 mmol) and acetic anhydride (0.5mL, 5mmol) were mixed together at 0° C. for 30 minutes, before being added toa solution2-(4-{[2-(hydroxyamino)-5-pyrirnidin-2-ylpentyl]sulfonyl}piperazin-1-yl)-5-(pyridin-2-ylethynyl)pyriridine(190 mg, 0.37 mmol) in tetrahydroflran (5 mL) and formic acid (1.8 mL)at 0° C. The reaction was allowed to reach room temperature and wasstirred for 45 minutes. The reaction was then evaporated in vacuo, andazeotroped with toluene (2×5 mL). The residue was then dissolved in MeOHand heated to 45° C. for one hour. The solution was evaporated in vacuo,and the residue triturated with Et₂O to give a white solid which wascollected by filtration, washed with Et₂O and dried in vacuo to givehydroxy{1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl}sulfonyl)methyl]4-pyrimidin-2-ylbutyl}formamideas a white solid. (64 mg, 32%).

[0194]¹H NMR (d6-DMSO@373k) δ 9.50 (br, s, 1 H), 8.64 (m, 2 H), 8.61 (m,3H), 8.17 (br, s, 1 H), 7.84 (ddd, 1 H), 7.58 (d, 1 H), 7.38 (m, 1 H),7.29 (dd, 1 H), 4.33 (br, s, 1 H), 3.96 (m, 4 H), 3.48 (dd, 1 H), 3.33(m, 4 H), 3.20 (dd, 1 H), 2.96 (m, 2 H), 1.74 (m, 4H).

[0195] MS (ESI): 537.07 (MH⁺)

[0196] The starting material was prepared as follows:

[0197] To a stirred solution of 5-iodo-2-piperazin-1-ylpyrimidine (7.0g, 24.1 mmol, CAS number 9584741-9) and triethylamine (10 mL, 72 mmol)in dichloromethane (70 mL) at 0° C. was added methanesulfonyl chloride(1.93 mL, 138 mmol) dropwise over 10 minutes. The reaction was thenstirred for 30 minutes at 0° C., before being allowed to warm to roomtemperature and stirred for an additional 30 minutes. The reaction wasthen quenched with water (70 mL) and the layers were separated. Theorganic phase was washed with water (100 mL) and the organics were dried(MgSO₄), filtered and evaporated in vacuo. The residue was thentriturated with ethyl acetate and the solid residue filtered and driedin vacuo to give 5-iodo-2-[4-(methylsulfonyl)piperazin-1-yl]pyrimidineas an off white solid (8.85g, 24 mmol, 99%).

[0198]¹H NMR (d6-DMSO): 8.54 (s, 2 H), 3.82 (m, 4 H), 3.17, (m, 4 H),2.87 (s, 3 H)

[0199] MS (ESI): 369.01 (MH⁺)

[0200] To a stirred suspension of5-iodo-2-[4-(methylsulfonyl)piperazin-1-yl]pyrimidine (5.52 g, 15 mmol)in THF (50 mL) at −10° C., was added dropwise a solution of LiHMDS inTHF (31.5 mL, 1.0M solution, 31.5 mmol). The resulting suspension wasstirred at −10° C. for 20 minutes before being treated with diethylchlorophosphate (2.24 mL, 15.5 mmol). The solution was then maintainedat −20° C. for 20 minutes before being treated with a solution of4-pyrimidin-2-ylbutanal (2.31 g, 15.5 mmol) in TBF (5 mL). The solutionwas then maintained at −20 ° C. for one hour before being quenched withsaturated aqueous ammonium chloride solution (50 mL). The layers wereseparated and the aqueous phase extracted with ethyl acetate (3×30 mL).The combined organic extracts were then dried, (MgSO₄), filtered andconcentrated in vacuo to give a brown soild which was purified by flashchromatography (silica gel, 20% to 50% to 100% EtOAc in hexanes) to give5-iodo-2-(4-{[(1E/Z)-5-pyrimidin-2-ylpent-1-enyl]sulfonyl}piperazin-1-yl)pyrimidineas a yellow solid (5.14 g, 68%, E:Z 1.7:1).

[0201]¹H NMR (CDCl₃): 8.58 (m, 2 H), 8.33 (m, 2 H), 7.18, (m, 1 H), 6.73(ddd, 1 H), 6.42 (ddd)*, 6.04 (d, 1H), 5.93 (d)*, 3.82 (m, 4H), 3.11 (m,4H), 2.94 (m, 2H), 2.63 (m)*, 2.29 (m, 2H), 1.94 (m, 2H)

[0202] minor geometrical isomer.

[0203] MS (ESI): 501.26 (MH⁺).

[0204] A stirred solution of5-iodo-2-(4-{[(lE/Z)-5-pyrimidin-2-ylpent-1-enyl]sulfonyl}piperazin-1-yl)pyrimidine,(500 mg, 1 mmol), Pd(PPh₃)₂Cl₂ (35 mg, 0.05 mmol), CuI (76 mg, 0.4mmol), triethylamine (0.4 mL, 3 mmol) and 2-ethynylpyridine (206 mg, 2mmol) in THF (20 mL) was stirred at room temperature for 3 hours. Themixture was then evaporated in vacuo, and the residue purified by flashchromatography (silica gel, 1% to 2% MeOH in EtOAc) to give5-(pyridin-2-ylethynyl)-2-(4-{[(1E/Z)-5-pyrimidin-2-ylpent-1-enyl]sulfonyl}piperazin-1-yl)pyrimidineas a white solid (224 mg, 47%).

[0205] MS (ESI): 476.39 (MH⁺)

[0206] To a stirred solution of give5-(pyridin-2-ylethynyl)-2-(4-{[(1E/Z)-5-pyrimidin-2-ylpent-1-enyl]sulfonyl}piperazin-1-yl)pyrimidine(220 mg, 0.46 mol) in THF (10 mL) at room temperature was added 50%aqueous hydroxylamine (2 mL) and the mixture stirred rapidly for 2hours. The reaction was quenched by the addition of saturated ammoniumchloride solution (5 mL) and the layers were then separated. The aqueousphase was then extracted with EtOAc (3×5 mL) and the combined organicextracts were then dried (MgSO₄), filtered and evaporated in vacuo. Thewhite solid obtained was then purified by flash chromatography (silicagel, 50% to 100% EtOAc in hexanes), to give 52-(4-{[2-(hydroxyamino)-5-pyrinidin-2-ylpentyl]sulfonyl}piperazin-1-yl)-5-(pyridin-2-ylethynyl)pyrimidineas a white solid (195 mg, 0.385 mmol, 83%).

[0207] MS (ESI): 507.06 (MH⁺)

EXAMPLE 10hydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl}sulfonyl)methyl]4-pyrimidin-2-ylbutyl}formamide

[0208]

[0209] The racemic mixture, prepared as in example 9, was separated bychiral HPLC (on a Merck Chiralpak AS-V column, 20 μm, 5 cm×25 cm, flowrate 50 mL/min, eluent=100% MeOH) to givehydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pymidin-2-yl]piperazin-1-yl}sulfonyl)methyl]4-pyrimidin-2-ylbutyl}formamideas a white solid.

[0210]¹H NMR (d6-DMSO@373k) δ 8 9.50 (br, s, 1 H), 8.64 (m, 2 H), 8.61(m, 3H), 8.17 (br, s, 1 H), 7.84 (ddd, 1 H), 7.58 (d, 1 H), 7.38 (m, 1H), 7.29 (dd, 1 H), 4.33 (br, s, 1 H), 3.96 (m, 4 H), 3.48 (dd, 1 H),3.33 (m, 4 H), 3.20 (dd, 1 H), 2.96 (m, 2 H), 1.74 (m, 4H).

[0211] MS (ESI): 537.07 (MH⁺)

EXAMPLE 11

[0212] The following compounds were also prepared.

Prepared using B R1 M + H method in example 4-F—Ph2-PyrimidinylCH2CH2CH2 554.40 9 4-Cl—Ph 2-PyrimidinylCH2CH2CH2 570.39 93-Pyridyl 2-PyrimidinylCH2CH2CH2 537.39 9 3-Pyridyl5-F-2-PyrimidinylCH2CH2 541.36 9 4-F—Ph 5-F-2-PyrimidinylCH2CH2 558.32 92-Pyridyl 5-F-2-PyrimidinylCH2CH2 541.37 9 4-Cl—Ph5-F-2-PyrimidinylCH2CH2 574.20 9

What we claim is:
 1. A compound of the formula I or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof

wherein B is selected from H, C₁₋₆alkyl, up to C12 cycloalkyl, up to C12aryl, and up to C12 heteroaryl; B is optionally substituted by up tothree groups independently selected from OH, NO₂, CF₃, CN, halogen,SC₁₋₄alkyl, SOC₁₋₄alkyl, SO₂C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₄alkoxy; L₁ and L₂are each independently selected from a direct bond and C₁₋₆alkyl; M₁,M₂, M₃, M₄ and M₅ are each independently selected from N and C; R1 isthe group -X-Y; X is C₁₋₆alkyl; Y is selected from up to C10 cycloalkyl,up to C10 aryl, and up to C10 heteroaryl; Y is optionally substituted byup to three groups independently selected from OH, NO₂, CF₃, CN,halogen, SC₁₋₄alkyl, SO₂C₁₋₄alkyl, SO₂C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₄alkoxy.2. A compound of the formula II or a pharmaceutically acceptable salt oran in vivo hydrolysable ester thereof

wherein B is selected from H, C₁₋₆alkyl, up to C12 cycloalkyl, up to C12aryl, and up to C12 heteroaryl; B is optionally substituted by up tothree groups independently selected from OH, NO₂, CF₃, CN, halogen,SC₁₋₄alkyl, SOC₁₋₄alkyl, SO₂C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₄alkoxy; L₁ and L₂are each independently selected from a direct bond and C₁₋₄alkyl; M₁,M₂, M₃, M₄ and M₅ are each independently selected from N and C; R1 isthe group -X-Y; X is C₁₋₆alkyl; Y is selected from up to C10 cycloalkyl,up to C10 aryl, and up to C10 heteroaryl; Y is optionally substituted byup to three groups independently selected from OH, NO₂, CF₃, CN,halogen, SC₁₋₄alkyl, SOC₁₋₄alkyl, SO₂C₁₋₄alkyl, C₁₋₄alkyl, C₁₋₄alkoxy.3. A compound as claimed in claim 1 or claim 2 or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof wherein B isselected from H, C₁₋₆alkyl, C6 aryl, and up to C6 heteroaryl.
 4. Acompound as claimed in claim 3 or a pharmaceutically acceptable salt oran in vivo hydrolysable ester thereof wherein B is selected from H,C₂₋₄alkyl, C6 aryl, and up to C6 heteroaryl.
 5. A compound as claimed inclaim 4 or a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof wherein B is up to C6 heteroaryl.
 6. A compound as claimedin claim 1 or claim 2 or claim 3 or a pharmaceutically acceptable saltor an in vivo hydrolysable ester thereof wherein B is not substituted orB is substituted by at least one group selected from CF₃, CN, halogen,C₁₋₄alkyl.
 7. A compound as claimed in claim 1 or claim 2 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof wherein at least one of L₁ and L₂ is a direct bond.
 8. Acompound as claimed in claim 7 or a pharmaceutically acceptable salt oran in vivo hydrolysable ester thereof wherein each of L₁ and L₂ is adirect bond.
 9. A compound as claimed in claim 1 or claim 2 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof wherein M₁ is N.
 10. A compound as claimed in claim 1 or claim 2or a pharmaceutically acceptable salt or an in vivo hydrolysable esterthereof wherein at least one of M₂, M₃, M₄, M₅ is C, and at least one ofM₂, M₃, M₄, M₅ is N.
 11. A compound as claimed in claim 10 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof wherein each of M₄ and M₅ is C, and at least one of M₂ and M₃ isN.
 12. A compound as claimed in claim 11 or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof wherein M₂ is Cor N and M₃ is N.
 13. A compound as claimed in claim 1 or claim 2 or apharmaceutically acceptable salt or an in vivo hydrolysable esterthereof wherein X is C₂₋₅alkyl.
 14. A compound as claimed in claim 13 ora pharmaceutically acceptable salt or an in vivo hydrolysable esterthereof wherein X is C₂₋₃alkyl.
 15. A compound as claimed in claim 1 orclaim 2 or a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof wherein Y is C6 aryl or C6 heteroaryl.
 16. A compound asclaimed in claim 15 or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof wherein Y is selected from phenyl, pyridyl,pyrimidinyl, or pyrazinyl.
 17. A compound as claimed in claim 1 or claim2 or claim 15 or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof wherein Y is not substituted or Y issubstituted by at least one halogen group.
 18. A compound as claimed inclaim 1 or a pharmaceutically acceptable salt or an in vivo hydrolysableester thereof wherein the compound of the formula I is as exemplifiedherein.
 19. A compound as claimed in claim 18 or a pharmaceuticallyacceptable salt or an in vivo hydrolysable ester thereof wherein thecompound is selected fromhydroxy{-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)methyl]-4-pyrimidin-2-ylbutyl}formamide,hydroxy[4-pyrimidin-2-yl-1-({[4-(5-{[5-(trifluoromethyl)pyridin-2-yl]ethynyl}pyridin-2-yl)piperazin-1-yl]sulfonyl}methyl)butyl]formamide,hydroxy{(3S)-3-phenyl-1-[({4-[4-(phenylethynyl)phenyl]piperazin-1-yl}sulfonyl)methyl]butyl}formamide,hydroxy[(3S)-3-phenyl-1-({[4-(4-ethynylphenyl)piperazin-1-yl]sulfonyl}methyl)butyl]formamide,hydroxy{4-pyrimidin-2-yl-1-[({4-[5-(thien-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)methyl]butyl}formamid,1-{[(4-{5-[(4-fluorophenyl)ethynyl]pyridin-2-yl}piperazin-1-yl)sulfonyl]methyl}-4-pyrimidin-2-ylbutyl(hydroxy)formamide,hydroxy{(1s)-1-[({4-[5-(pyridin-2-ylethynyl)pyridin-2-yl]piperazin-1-yl}sulfonyl)methyl]+pyrimidin-2-ylbutyl}formamide,hydroxy{1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl}sulfonyl)methyl]4-pyrimidin-2-ylbutyl}formamide,andhydroxy{(1S)-1-[({4-[5-(pyridin-2-ylethynyl)pyrimidin-2-yl]piperazin-1-yl}sulfonyl)methyl]-4-pyrimidin-2-ylbutyl}formamide.20. A pharmaceutical composition which comprises a compound of theformula I as claimed in claim 1 or a compound of the formula II asclaimed in claim 2 or a pharmaceutically acceptable salt or an in vivohydrolysable ester thereof and a pharmaceutically acceptable carrier.21. A compound of the formula I as claimed in claim 1 or a compound ofthe formula II as claimed in claim 2 or a pharmaceutically acceptablesalt or in vivo hydrolysable ester thereof for use in a method oftherapeutic treatment of the human or animal body.
 22. A compound of theformula I as claimed in claim 1 or a compound of the formula II asclaimed in claim 2 or a pharmaceutically acceptable salt or in vivohydrolysable ester thereof for use as a therapeutic agent.
 23. A methodof treating a metalloproteinase mediated disease or condition whichcomprises administering to a warm-blooded animal a therapeuticallyeffective amount of a compound of the formula I as claimed in claim 1 ora compound of the formula II as claimed in claim 2 or a pharmaceuticallyacceptable salt or in vivo hydrolysable ester thereof.
 24. A method oftreating a metalloproteinase mediated disease or condition as claimed inclaim 23 which comprises treating a disease or condition mediated byMMP13.
 25. The use of a compound of the formula I as claimed in claim 1or a compound of the formula II as claimed in claim 2 or apharmaceutically acceptable salt or in vivo hydrolysable precursorthereof in the preparation of a medicament for use in the treatment of adisease or condition mediated by one or more metalloproteinase enzymes.26. The use of a compound of the formula I as claimed in claim 1 or acompound of the formula II as claimed in claim 2 or a pharmaceuticallyacceptable salt or in vivo hydrolysable precursor thereof in thepreparation of a medicament for use in the treatment of arthritis.